Multiplex labeling of cells for analysis of biological samples, e.g., mixed cell populations, has been described. However, known methods have limitations which are dictated by the finite number of fluorescence emission colors of known organic fluorophores which can be squeezed into the visible, near-ultraviolet (UV), near-infrared (IR) spectral regions in which conventional measurements are made, e.g., by flow cytometry. These limitations include the widths of emission bands, the spectral overlap between these emission bands, and the excitation wavelength requirements.
Two examples of labels for cells are CdSe core nanoparticles which have been used for biological staining and observation with a fluorescence microscope [Bruchez J. M. et al., Science 281, 2013 (1998) and Chan, W. C. W. and Nie, S., Science 281, 2016 (1998)].
As the upper limit in the number of usable colors was reached, other methods, based on fluorescence intensity differences have been developed. For example, mutually exclusive pairs of targeted white blood cell populations with widely different, intrinsic numbers of receptors per cell can be labeled by a single color marker and analyzed by flow cytometry [U.S. Pat. No. 5,538,855].
Several non-radioactive gene probes, oligos with attached fluorescent dye that hybridize or bind to sample DNA have been described [L. M. Smith et al., Nature, 321:674-679 (1986) and L. M. Smith et al, Nucl. Acids Res., 13:2399-2412 (1985)] and are being used for labeling of biological samples. Automated DNA sequencers use four fluorescent dyes with non-overlapping emission bands, one per nucleotide base. However, electrophoretic mobilities of the fluorescent dye-oligo primer conjugates need to be similar for all four conjugates. Also the molecular weight of the conjugates cannot be too high, otherwise they will not move through the polyacrylamide or agarose gel used in the electrophoresis.
The need for increased sensitivity of probes used in automated analysis by attaching multiple marker molecules per oligonucleotide primer were recognized as early as 1986 [L. M. Smith et al, cited above]. However, only a limited degree of fluorescence enhancement has been possible for dye-oligo conjugates that are constrained to low molecular weight for separation by gel electrophoresis.
Aminodextrans have been used as reducing and/or protective agents in the preparation or coating of monodispersed colloidal dispersions of magnetic ferrite [U.S. Pat. No. 5,240,640], metal [U.S. Pat. No. 5,248,772], polystyrene [U.S. Pat. No. 5,466,609; U.S. Pat. No. 5,707,877; U.S. Pat. No. 5,639,620; U.S. Pat. No. 5,776,706], and polystyrene-metal [U.S. Pat. No. 5,552,086; U.S. Pat. No. 5,527,713] particles. Aminodextran of sufficiently large molecular weight can accommodate multiple protein molecules. Complexes containing such aminodextrans conjugated to a ligand and a selected fluorescent marker or label have been described. [See, Smith, C., et al, "Detection of Low-Density Surface Markers Using Novel Amplified Fluorochrome-Conjugated Antibodies", Cytometry, Suppl. 9, p. 56, presented at XIX Congress of International Society for Analytical Cytology, Mar. 3-7, 1998; R. Mylvaganam, et al., "Seven Markers, Four Colors, Single Laser Flow Cytometry Using Amplified Fluorochrome Conjugated Antibodies", Cytometry, Suppl. 9, p. 117 (1998), as presented at XIX Congress of International Society for Analytical Cytology, Mar. 3-7, 1998.] However, there continues to be a need for probes which permit increased assay sensitivity, by providing narrower fluorescence bandwidths and enhanced intensities, decreased probe size and increased probe stability.